Abstract 6886: Orion™: 17-plex single-step stain and imaging of normal lung section adjacent to non-small cell lung carcinoma

Abstract Background: The ability to image tissue microenvironments (TME) at high-plex over a whole slide without requiring multiple staining steps allows for unprecedented ability to study tissue architecture and molecular mechanisms of immune and disease processes. Here we investigate a sample of normal lung section adjacent to non-small cell lung carcinoma using whole slide, single-step stain and imaging at single-cell resolution. Methods: These images profiled a whole slide tissue section of normal lung section adjacent to non-small cell lung carcinoma, stained with a 17-plex immuno-oncology biomarker panel. In this profile, we designed a high-plex panel of 17 biomarkers where the tissue autofluorescence was imaged and isolated as an additional fluorescence channel. All markers were stained in a single staining procedure and imaged in a single scan. Whole slide spatial staining and imaging was conducted on the Orion spatial biology platform. The full protocol is quick and simple, using standard histology tools: •Mount sections on glass slides•De-paraffinize and perform antigen retrieval•Quench autofluorescence•Stain slides with a panel of ArgoFluor™ conjugated antibodies•Coverslip with ArgoFluor Mounting Medium and cure overnight•Image whole slides at 20X magnification using the Orion instrument•Process to ome. TIFF and analyze. Results: Data revealed intact normal lung alveolar architecture, with scattered immune cell infiltrates. Multiplexed imaging revealed epithelial cells, which make up alveoli, and endothelial vasculature as well as immune cells infiltrates in the alveolar space. Various immune cell populations were observed to be present (T cells, B cells, macrophages) and immune cell activation could be distinguished using PCNA and/or Ki-67 markers. Various T cell subsets could also be identified among the infiltrate using markers for CD4, CD8 and T-regulatory marker FOXP3. Through the use of multiplex staining, immune cell activity could also be characterized with respect to bronchiolar, artery, and capillary tissue architecture. Conclusions: High-quality subcellular imaging of TME can lead to greater spatial investigation and more in-depth understanding of immune function in normal and diseased tissue. Here, multiplexed imaging identified distinct immune cell collections among cancer-adjacent normal lung tissue, providing validation of instrumentation and methods to obtain benchmarks for spatial quantitation of immune cells in control regions adjacent to cancer tissue. Citation Format: Selena Larkin, Tad George, Eric Kaldjian. Orion™: 17-plex single-step stain and imaging of normal lung section adjacent to non-small cell lung carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6886.